Background: The peracetic acid (PAA) has antimicrobial activity against bacteria and fungi including spores and is envirosafe. Despite its widespread use in food industry and effluent treatment, it is not widely used in hospitals. The present work was aimed to find out its efficacy against hospital pathogens, bacterial and fungal spores and mycobacteria.Methods: Multidrug-resistant, wild hospital isolates of Gram-positive, Gram-negative bacteria, tough organisms such as Mycobacterium fortuitum, Mycobacterium tuberculosis and Candida albicans and spores of Bacillus subtilis, Clostridium perfringens and Aspergillus niger were checked by modified Kelsey-Sykes suspension test. For vegetative bacteria, exposure time was 1 min. For bacterial and fungal spores, Candida and Mycobacteria, exposure time varied from 10 to 30 min.Results: More than 5-log reduction was seen for vegetative bacteria just after 1 min exposure to PAA. Ten minute exposure to PAA could inactivate 99.5% bacterial and fungal spores. Mycobacteria were inactivated within 10 min of exposure to PAA. PAA rapidly inactivates pathogenic bacteria within 1 min and inactivates mycobacteria and fungi within 10 min and sterilises spores within 30 min and remains active in the presence of proteins.Conclusions: It is economic, eco-friendly and deserves major share in hospital disinfection.

There has been ever-increasing use of disinfectants in hospital practice for surface disinfection and to reduce hospital-acquired infections. An ideal disinfectant has to have rapid microcidal activity, non-interference of other factors such proteins, pH, temperature, hardness of water, adequate stability at in-use dilution, wide spectrum of coverage against pathogens including bacteria, mycobacteria, fungi, fungal and bacterial spores and viruses.[1],[2],[3] Further, it should be safe for the environment and users, microbes should not develop resistance for it and cost should be affordable.[2] Hypochlorite though widely used in hospitals has limitations such as poor stability (diluted solution stable for few hours) and activity grossly reduced in the presence of organic matter.[4],[5],[6] Aldehydes are not eco-friendly and are not considered safe for the use.[7],[8],[9],[10] In fact, European Union has recommended the withdrawal of aldehyde from the European market. Even chloroxylenol is not considered safe by international agencies and has been suggested to be withdrawn.[10] Chlorhexidine is popularly used as antiseptic/disinfectants but does not cover spores, mycobacteria and some other microbial agents at the 'in-use dilution'.

The peracetic acid (PAA) has more advantages than the above-mentioned disinfectants and hence is closer to the ideal disinfectants. PAA is a disinfectant that has desirable properties of hydrogen peroxide, i.e., broad spectrum activity against microorganisms, lack of harmful decomposition product and infinite water solubility.[7] Its chemical formula is acetic acid plus an extra oxygen atom.[7] This extra oxygen atom is highly reactive and reacts with most cellular components and resulting in cell death.[11] On decomposition, PAA forms acetic acid (vinegar) and oxygen, rendering it non-toxic and environmentally safe.[12] Until now, no microbial resistance to PAA has been reported.[13]

PAA has greater lipid solubility than hydrogen peroxide and is free from deactivation by catalase and peroxidase enzymes.[11] PAA can be used over a wide temperature range (0–40°C) and is free of protein interference.[14] It is efficient over a wide spectrum of pH (3–7.5) and can be used with hard water also.[15] PAA has also excellent sporicidal activity.[16],[17] It has been accepted worldwide in the food processing and beverage industries as being ideal for clean-in-place system and as a disinfectant for fruits, vegetables, meat and eggs.[18] It is also used as a sanitizer in the pharmaceutical and cosmetic industries.[19],[20]

PAA has been proposed as a disinfectant for urban sewage either alone [21] or in combination with ultraviolet irradiation.[22] The United States Environmental Protection Agency first registered PAA as an antimicrobial in 1985 for indoor use on hard surfaces.[23] PAA is not widely used in Indian hospital practice. It was therefore aimed to find out efficacy of PAA against medically important bacterial and fungal pathogens, spores and mycobacteria.

Subcultures of Gram-positive and Gram-negative bacteria were made on nutrient agar (Hi-Media, India). B. subtilis was sporulated and the spore suspension heated at 70°C for 30 min to inactivate vegetative forms. C. perfringens grown in Robertson cooked meat medium for 7 days and the spores heated at 70°C for 30 min to inactivate vegetative forms. The wild isolate of C. perfringens was selected because of its ability to form large number of spores. C. albicans was subcultured on Sabouraud agar slant (Hi-Media, India) and incubated at 30°C for 48 h. A. nige r spores were from subculture made on Sabouraud agar slant incubated at 30°C for 5 days. The spores were harvested in 5 ml sterile normal saline. M. fortuitum and M. tuberculosis were subcultured on Lowenstein Jensen medium (in-house prepared) and incubated at 37°C for 48 h and 4 weeks, respectively.

Experiment to check the effect of peracetic acid on the tough organisms such as Candida albicans, Mycobacteria fortuitum and Mycobacteria tuberculosis

Candida was grown on Sabouraud agar slants and M. fortuitum and M. tuberculosis on L.J. medium slants. Growth was harvested in normal saline and density adjusted to 0.5 McFarland standards (approximately 108 cells/mL). Exposure time was 10 min, 20 min and 30 min. After exposure time, 2 μl suspensions spread on slants. Sabouraud agar slants were used for viable count of Candida and the colonies counted after 48 h at room temperature while M. fortuitum inoculated on L.J. slants incubated at 37°C for 48 h. For M. tuberculosis, L.J. slants were incubated at 37°C for 4–6 weeks. For viable counts, /102, /103 and /104 dilutions were used [Table 3].

Sterilisation is a process, in which the complete destruction or elimination of all living microorganisms including their spores, accomplished by physical method (dry or moist heat), chemical agents (ethylene oxide, aldehydes, alcohol, PAA), radiation (ultraviolet, cathode) or mechanical method (filtration). Biological indicators for sterilisation are based on viability reduction by five logs (European Pharmacopoeia, 2005).[24]

The viability of common bacterial pathogens including MDR ESBL E. coli, Klebsiella, MRSA, PAN-resistant Pseudomonas and Acinetobacter baumannii was reduced by more than five logs on exposure to PAA just for 1 min [Table 1].

More than 5-log reduction was also seen for B. subtilis and C. perfringens spores at the end of 30 min and thus sterilisation of spores was evident after 30 min exposure [Table 2].

A. niger spores and Mycobacteria such as M. fortuitum and M. tuberculosis were also inactivated (4–5-log reduction) at 10 min exposure to PAA [Table 3].

The experimental data in the present study clearly document very high bactericidal activity (>5-log reduction) of PAA against both Gram-positive and Gram-negative bacteria. It needs to be mentioned that the MDR and tough isolates from the hospitalised cases including MDR Pseudomonas, and Acinetobacter, ESBL E. coli and Klebsiella, MRSA, VRE and all were efficiently inactivated after just 1 min exposure to PAA. The time required for vegetative pathogenic bacteria could be <1 min. However, experimental design for the viability assessment at time point <1 min appeared practically difficult. Hence, the challenged suspensions were checked for viability after 1 min exposure by neutralisation of the PAA. For neutralisation, 100 times volume of neutraliser containing 0.5% sodium thiosulphate was used. The instant addition of large volume of neutraliser could give sufficient time for viable count study.

Discussion

The rapid and broad-spectrum activity appears to be due to extra oxygen atom present with the acetic acid.[11],[12] Further, after release of oxygen atom decomposition to acetic acid and oxygen makes the PAA environmentally safe disinfectant.[11] PAA has greater lipid solubility and is free from deactivation by catalase and peroxidase enzyme [12] and has activity over a wide temperature (0–40°C).[14] PAA can also be used with hard water over a wide pH range from 3 to 7.5.[17],[19] The important part is its effectiveness in the presence of organic matter such as proteinaceous substance and the point scores advantage over hypochlorite and iodine.[13] In the present study, the activity was checked in the presence of 5% serum and confirms the above-mentioned view.

Bacterial spores are tough and offer resistance to most of the chemical disinfectants. Aldehydes require several hours (4–24 h) to kill bacterial spores, but the efficient sporicidal activity was evident in 30 min of exposure to PAA and thus the agent qualifies even as sterilising agent at 30 min exposure (more than 5-log reduction in viability). The PAA also showed very good activity against fungi such as C. albicans and Aspergillus spores. The tough mycobacteria were also inactivated after 10 min exposure to PAA. The virucidal effect was not checked in the present study, but 300 ppm of PAA is reported to inactivate virus.[24]

The U.S. Food and Drug Administration and the Centers for Disease Control and Prevention have accepted PAA as safe.[25] In fact, PAA is used in food industry for disinfection purpose [26] effluent treatment [12],[27] and Pharma industry, in hospitals for sterilisation and high-level disinfection of medical equipment such as dental equipment, endoscopes and arthroscopes.[28] At our centre, we have been using PAA instead of aldehyde preparation in operation theatre cleaning since 2007; PAA has replaced formaldehyde for dialysis membranes in Dialysis Unit. Diluted PAA is more stable (>10 days) compared to 6–8 h for diluted hypochlorite.[11] Almost all Western countries have stopped the use of aldehydes in the hospital practice; this is because of the unfriendly and likely carcinogenic effect of the aldehydes.[10]

Conclusions

PAA is rapid in action, destroys vegetative bacteria including mycobacteria, inactivates bacterial and fungal spores, under laboratory conditions and is eco-friendly.

Acknowledgement

We are thankful to the management, Choithram Hospital and Research Centre, Indore, India, for the research facility support.

II Acts adopted under the EC Treaty/Euratom Treaty whose publication is obligatory: Commission decision of 14 October 2008 concerning the non-inclusion of certain substances in Annex I, IA or IB to Directive 98/8/EC of the European Parliament and of the Council concerning the placing of biocidal products on the market. Official Journal of the European Union 2008;51(L281):16-29.